Effects of transfection with acidic fibroblast growth factor by electroporation on skeletal muscle satellite cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Previous studies have shown that a certain dose of acidic fibroblast growth factor can promote skeletal muscle satelite cel proliferationin vitro. OBJECTIVE:To investigate the effects of transfection with acidic fibroblast growth factor by electroporation on growth, proliferation and differentiation of skeletal muscle satelite cels. METHODS: Skeletal muscle satelite cels were cultured and purified, and then transfected with plasmid pSectag-GFP-aFGF by electroporation. The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was calculated. After transfection, cel cycle was analyzed by flow cytometry to draw the growth curve of skeletal muscle satelite cels. Western blot assay was employed to measure protein level of acidic fibroblast growth factor. RESULTS AND CONCLUSION: (1) Immunocytochemistry detection The skeletal muscle satelite cels were positive for a-sarcomeric actin. (2) Transfection efficiency At 12 hours after transfection with pSectag-aFGF, several cels showed green fluorescence, and the green fluorescent expression reached the peak at 72-96 hours after transfection with a positive rate of about 90%. (3) Cel cycle After electrotransfection, the proportion of cels at S phase in the electroporation group was higher than that in the control group (P < 0.05). (4) Cel growth curve At 3 days after electrotransfection, the cels entered logarithmic growth phase but the proliferation slowed down at 5 days. (5) Differentiation capacity There were fewer myotubes and aging cels in the electroporation group than the control group. (6) Western blot assay Acidic fibroblast growth factor protein was highly expressed in the cels transfected with target gene detected by western blot assay. These findings indicate that by using electroporation method, acidic fibroblast growth factor can be transferred into skeletal muscle satelite cels and have a high-efficiency and long-term expression, which can promote the proliferation of skeletal muscle satelite cels and inhibit formation of myotubes.